We have previously reported that mice ablated for nonmuscle myosin (NM) II-B (B-/B-) have reduced numbers of cardiac myocytes and an abnormal accumulation of bi-nucleated myocytes in embryonic hearts due to a failure in cytokinesis. The aorta and pulmonary artery of B-/B- mouse hearts both originate from the right ventricle (DORV). Here we report on mice which carry a motor-impaired NM II-B, where Arg709 is mutated to Cys in the myosin heavy chain. No abnormal accumulation of bi-nucleated cardiac myocytes is seen in these BC/BC mice, suggesting that mutant II-B can participate in cardiac myocyte cytokinesis, although heavy meromyosin from the mutant II-B is incapable of propelling actin filaments in vitro. However, BC/BC hearts still show a thin, less compact myocardium with significantly reduced numbers of cardiac myocytes. The proliferation rate of cardiac myocytes was also reduced, determined by BrdU labelling. BC/BC hearts show a disorganized epicardium, supporting the idea that these cells provide a proliferation signal to the myocardium. Similar to B-/B- mice, BC/BC mice also develop DORV, indicating that NM II-B and its motor activity are essential for normal development of the cardiac outflow tract (OFT). Ablation of NM II-C does not result in DORV and replacement of II-B with II-A failed to rescue DORV suggesting that NM II-B plays an isoform-specific role in OFT development. Cardiac myocytes of the OFT exhibit polarized migration into the cushion tissues in wild-type mice. Polarized migration is not seen in NM II-B ablated and BC/BC mice. Thus, it appears that NM II-B is required for polarized migration of cardiac myocytes in the developing OFT and that failure in migration of the myocytes is the cause of DORV in NM II-B ablated and mutated mice.